L-selectin mediates the initial adhesion of lymphocytes to high endothelial venules (HEV) in lymph nodes during the process of lymphocyte recirculation. It also functions in leukocyte-leukocyte and leukocyte-endothelial interactions that occur during trafficking of leukocytes into inflammatory sites in both acute and chronic settings. L-selectin functions as a lectin-like receptor in recognizing a discrete set of HEV-ligands including GlyCAM-1, CD34, podocalyxin, Sgp200 and MAdCAM-1. These ligands are sulfated, fucosylated and sialylated, and all three of these modifications are required for optimal recognition by L-selectin. In addition, these glycoproteins are recognized by the monoclonal antibodies (MECA 79 and G72), which bind to sulfated substructures that are essential for their ligand activity. A detailed carbohydrate analysis of GlyCAM-1 has revealed that specific sulfation modifications of sialyl Lewis X are found: Gal-6-sulfation and N- acetylglucosamine-6-sulfation. This proposal is directed at the identification of the sulfotransferases, which catalyze these specific sulfation modifications of the HEV-associated ligands. One of the few cloned sulfotransferases that modifies carbohydrate chains is the chick chondroitin 6/keratan sulfate sulfotransferase (C6ST/KSST). This enzyme is capable of catalyzing the addition of sulfate to the 6-position of Gal in simple acceptor structures. We gave recently cloned 3 human cDNAs (GST 1, 2, and 3) which are highly homologous to the chicken C6ST/KSST. One of these (GST 3) is selectively expressed in the endothelial cells of HEV. The specific aims of the present proposal are: 1)To determine whether expressed proteins corresponding to the newly cloned GSTs (especially GST 3) catalyze the appropriate addition of sulfate moieties to model carbohydrate acceptors and to the carbohydrate chains of GlyCAM-1; 2) To identify the sulfated carbohydrate epitope recognized by MECA 79; and 3) To obtain cDNAs encoding HEC sulfotransferases by expression cloning techniques using the sulfation-dependent mAbs (G72 and MECA 79) and L-selectin. Understanding these enzymes has important biomedical implications, because the regulation of these enzymes provides a potential mechanism to control the expression of functional ligands for L-selectin, and thereby to control leukocyte trafficking into lymphoid organs and inflammatory sites.